HCRP1, a novel gene that is downregulated in hepatocellular carcinoma, encodes a growth-inhibitory protein

One of the most frequent allelic deletions in hepatocellular carcinoma (HCC) has been found at chromosome 8p21-23. We reported here the identification and characterization of a novel gene for a hepatocellular carcinoma related protein 1 (HCRP1) localized at 8p22, which was isolated by positional candidate cloning. The expression of the gene for HCRP1 was most abundant in normal human liver tissue and significantly reduced or undetected in HCC tissues. The analysis of subcellular distribution showed that HCRP1 diffused in the cytoplasm with a significant fraction accumulated in the nuclei. After introduction of the sense and antisense cDNA of HCRP1 into HCC cell line SMMC-7721, we observed that the overexpression of HCRP1 significantly inhibited both anchorage-dependent and anchorage-independent cell growth in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of HCRP1 gene in the other HCC cell line BEL-7404 resulted in the cell growth greatly enhanced. Moreover, reduction of the HCRP1 gene expression could also elevate the invasive ability of BEL-7404 cells. Our results strongly suggest that HCRP1 might be a growth inhibitory protein and associated with decreasing the invasion of HCC cells.     

Dermatopontin is expressed in human liver and is downregulated in hepatocellular carcinoma

Dermatopontin (DPT) was recently found as a downstream target of vitamin D receptor, which is a key molecule in the 1,25-dihydroxy-vitamin D₃ anti-hepatoma proliferation pathway. MCTx-1 from Millepora, a homolog of DPT, is identified as a cytotoxin towards leukemia cells. The aim of this study was to analyze DPT expression in hepatocellular carcinoma (HCC) based on the analysis for DPT gene in normal tissues in order to estimate its function in the progression of HCC. DPT mRNA expression was analyzed in normal tissues and HCC cell lines by RT-PCR, and in HCC tissue by RT-PCR and real-time PCR. Its protein was examined in HCC tissues by Western blot and immunohistochemistry assays. Meanwhile, transforming growth factor-β1 (TGF-β1) that is closely associated with HCC and DPT was observed by immunohistochemistry in HCC tissues. The results showed that DPT mRNA was strongly expressed in human fetal and adult liver, kidney, and spleen, weakly in ovary and heart, and absent in other tissues and HCC cell lines examined. Its mRNA was significantly downregulated in HCC tissues, while its protein was weakly expressed in tumor compared with non-tumor. DPT is located mainly in the cytoplasm of several cell types in the liver; it has been identified also in the extra-cellular matrix of the skin. TGF-β1 was observed in extensive tumor tissue of HCC. This fact suggests that DPT can play various roles in different tissues and might be a molecule related to carcinogenesis and the progression of HCC via possible interaction with TGF-β1 and other potential mechanisms.     

Ibulocydine is a novel prodrug Cdk inhibitor that effectively induces apoptosis in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC.     

A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa

Human and pig cDNAs for a novel stomach protein, the product of a gene expressed at high levels specifically in cells of the antrum mucosa, have been characterized. The general exon/intron structure of the genomic DNA is conserved in humans and mice. The predicted protein sequences of the human and mouse mRNAs contain 185 and 184 amino acids, respectively. The protein isolated from pig antral extracts has an NH2 terminus consistent with cleavage of a 20-amino acid signal peptide. Human cDNA was expressed in E. coli to generate a protein antigen for antibody production. The antibodies detected polypeptides of approximately 18 kDa in antrum extracts from all mammalian species tested. Immunocytochemistry located antrum mucosal protein (AMP)-18 to surface mucosal cells of the mouse antrum and, specifically, to secretion granules, suggesting that it is cosecreted with mucins. Antrum extracts and recombinant human AMP-18 exhibit growth-promoting activity on epithelial cells that can be blocked by the specific antisera. We suggest that AMP-18 is a "gastrokine" that maintains the integrity of the gastric mucosal epithelium.     

hZG16, a novel human secreted protein expressed in liver, was down-regulated in hepatocellular carcinoma

Here we reported a novel human secreted protein named as hZG16, with a Jacalin domain. Evolution analysis through comparing with the orthologs of other organisms suggested that ZG16 is a conserved gene under the purifying selection (d(N)/d(s)<1) in the evolution. Interestingly, Northern and dot blot analyses showed that hZG16 were highly expressed in adult liver, not in fetal liver, and moderately in gut, including jejunum, ileum, and colon, in which the tissue expression pattern of hZG16 was significantly dissimilar to that of mouse and rat orthologs that were uniquely expressed in spleen and pancreas, respectively. Unexpectedly, hZG16 was markedly down-regulated in hepatocellular carcinoma (HCC) as indicated by RT-PCR, Northern blot analysis and immunohistochemistry staining. However, the tunicamicin treatment and pulse-chase experiments showed that hZG16 protein had a similar molecular function with rZG16 that take part in glycoproteins' secretion in a bus mode.     

ILPIP,a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis

We have previously described a new aspect of the Inhibitor of Apoptosis (IAP) family of proteins anti-apoptotic activity that involves the TAK1/JNK1 signal transduction pathway (1,2). Our findings suggest the existence of a novel mechanism that regulates the anti-apoptotic activity of IAPs that is separate from caspase inhibition but instead involves TAK1-mediated activation of JNK1. In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we isolated by yeast two-hybrid screening a novel X chromosome-linked IAP (XIAP)-interacting protein that we called ILPIP (hILP-Interacting Protein). Whereas ILPIP moderately activates JNK family members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1, JNK2, and JNK3. The expression of a catalytically inactive mutant of TAK1 blocked XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling pathway. ILPIP moderately protects against interleukin-1beta converting enzyme- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of XIAP. In vivo co-precipitation experiments show that both ILPIP and XIAP interact with TAK1 and tumor necrosis factor receptor-associated factor 6. Finally, expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation, further supporting the idea that XIAP protection against apoptosis is achieved by two separate mechanisms: one requiring JNK1 activation and a second involving caspase inhibition.     

From mosquito to man: Identification of a novel protein kinase, HsHPK, which is highly expressed in human hepatoma tissues

Protein kinases play an important role in the signaling pathway of growth factors in most of the higher organisms. During the study of protein kinase profiles of mosquitoes using RT-PCR and degenerate primers for consensus catalytic domain motifs to amplify protein kinase genes, we have noticed that a novel mosquito kinase, AaPK-38, shares a stretch of amino acids identical to the corresponding domain in Tousled gene ofArabidopsis thaliana that is required for leaf and flower development. A 2.1-kb cDNA encoding human HsHPK gene, which is a homolog of AaPK-38, was isolated from human testis cDNA library. This cDNA contains an open reading frame of 563 amino acids, with a complete kinase domain in its carboxyl terminus. The expressed Flag-tagged HsHPK was shown to have kinase activity based on in vitro autophosphorylation. Northern blot analysis revealed that human HsHPK mRNA is most abundant in testes, much less in heart and skeletal muscle and almost undetectable in liver and lung. Finally, we found that the expression of HsHPK in 4 out of 6 human hepatoma tissues is much higher than that in the adjacent normal counterpart. This result suggests HsHPK may play a role in the development of human hepatoma.     

ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas

BACKGROUND: Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways. RESULTS: Here we report the characterization of ML-IAP, a novel human IAP that contains a single BIR and RING finger motif. ML-IAP is a powerful inhibitor of apoptosis induced by death receptors and chemotherapeutic agents, probably functioning as a direct inhibitor of downstream effector caspases. Modeling studies of the structure of the BIR domain revealed it to closely resemble the fold determined for the BIR2 domain of X-IAP. Deletion and mutational analysis demonstrated that integrity of the BIR domain was required for anti-apoptotic function. Tissue survey analysis showed expression in a number of embryonic tissues and tumor cell lines. In particular, the majority of melanoma cell lines expressed high levels of ML-IAP in contrast to primary melanocytes, which expressed undetectable levels. These melanoma cells were significantly more resistant to drug-induced apoptosis. CONCLUSIONS: ML-IAP, a novel human IAP, inhibits apoptosis induced by death receptors and chemotherapeutic agents. The BIR of ML-IAP possesses an evolutionarily conserved fold that is necessary for anti-apoptotic activity. Elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the pathogenesis of this malignancy.     

Targeting A20 enhances TRAIL-induced apoptosis in hepatocellular carcinoma cells

A20 was initially identified as a primary gene product following TNF α treatment in human umbilical vein endothelial cells. Increased A20 expression is associated with tumorigenesis in many cancers, whereas the loss of A20 function is linked to lymphoma. It has been reported that A20 protects cells from TRAIL-induced apoptosis; however, the mechanism by which A20 is involved is still largely unknown. Our results indicate that TRAIL induces the hepatocellular carcinoma apoptosis associated with A20 knockdown in a concentration-dependent manner. TRAIL-induced apoptosis requires p18 caspase-8 activation, and, the activation of caspase-8 is at least in part, due to the direct cleavage of RIP1 by A20 knockdown. These findings suggest that A20 modulates the sensitivity to TRAIL by RIP1 ubiquitination, thereby repressing the recruitment and activation of pro-caspase-8 into the active form caspase-8. Thus, our study suggests that A20 protects against TRAIL-induced apoptosis through the regulation of RIP1 ubiquitination.     

The human LIS1 is downregulated in hepatocellular carcinoma and plays a tumor suppressor function

The human lissencephaly-1 gene (LIS1) is a disease gene responsible for Miller–Dieker lissencephaly syndrome (MDL). LIS1 gene is located in the region of chromosome 17p13.3 that is frequency deleted in MDL patients and in human liver cancer cells. However, the expression and significance of LIS1 in liver cancer remain unknown. Here, we investigated the expression of LIS1 in hepatocellular carcinoma (HCC) tissues by real-time PCR, Western blot, and immunohistochemistry. The results indicated that the mRNA and protein levels of LIS1 were downregulated in about 70% of HCC tissues, and this downregulation was significantly associated with tumor progression. Functional studies showed that the reduction of LIS1 expression in the normal human liver cell line QSG7701 or the mouse fibroblast cell line NIH3T3 by shRNA resulted in colony formation in soft agar and xenograft tumor formation in nude mice, demonstrating that a decrease in the LIS1 level can promote the oncogenic transformation of cells. We also observed that the phenotypes of LIS1-knockdown cells displayed various defective mitotic structures, suggesting that the mechanism by which reduced LIS1 levels results in tumorigenesis is associated with its role in mitosis. Furthermore, we demonstrated that ectopic expression of LIS1 could significantly inhibit HCC cell proliferation and colony formation. Our results suggest that LIS1 plays a potential tumor suppressor role in the development and progression of HCC.     

A novel serum chitinase that is expressed in bovine liver

Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.     

FHL5, a novel actin-binding protein, is highly expressed in eel gill pillar cells and responds to wall tension

Supporting evidence for the contractile nature of fish branchial pillar cells was provided by demonstrating the presence of actin fibers and a novel four-and-a-half LIM (FHL) protein in which expression is specific for contractile tissues and sensitive to the tension applied to the pillar cell. When eel gill sections were stained with rhodamine-phalloidin, a selective fluorescent probe for fibrous actin, a strong bundle-like staining was observed around collagen columns in pillar cells, suggesting the presence of abundant actin fibers. A cDNA clone encoding a novel member of the actin-binding FHL family, FHL5, was isolated from a subtracted cDNA library of eel gill. Northern analysis revealed that FHL5 mRNA is highly expressed only in gills, heart, and skeletal muscle. In gills, FHL5 was found to be confined to pillar cells by immunohistochemistry. Confocal fluorescence microscopy showed that FHL5 is present in both cytosol and nucleus; within the cytosol, a large portion of FHL5 is colocalized with the phalloidin-positive actin bundles. Furthermore, transfection of myogenic C2C12 cells with FHL5 cDNA demonstrated, in addition to its interaction with actin stress fibers, a nuclear shuttling activity of FHL5. The mRNA and protein levels were found to be elevated on 1) transfer of eels from seawater to freshwater, 2) volume expansion by infusion of isotonic dextran-saline, and 3) constriction of gill vasculature by bolus injection of endothelin-1. These results suggest contractile nature of pillar cells and a role of FHL5 in maintaining the integrity and regulating the dynamics of pillar cells.     

Characterization of human LNX, a novel ligand of Numb protein X that is downregulated in human gliomas

Gliomas are major tumors of the central nervous system with a wide spectrum of different tumor types. Ligand of Numb protein X (LNX) is PDZ domain containing protein that interacts with cell fate determinant Numb. cDNA microarray analysis was used to determine the expression of 13,939 genes in a set of 18 gliomas. It showed that human LNX was downregulated in 100% of gliomas including low- and high-grade ones, which was confirmed by Northern blot. In situ hybridization analysis revealed that LNX was lowly expressed in cytoplasm of glioma cells. Thus, LNX might act as a diagnostic marker and a potential therapeutic target for glioma. Two-hybrid screen in yeast was used to identify human LNX interacting proteins important for LNX function. It showed that human LNX interacted with Ski interacting protein (SKIP) via PDZ domains. The co-immunoprecipitation results suggested that LNX interacted with SKIP in HEK293 cells. LNX could affect the subcellular localization of Numb, which indicated that LNX might function as a molecular anchor that localized Numb to the subcellular site of its interaction with Notch. The presence of multiple protein binding domains involved in signal transduction and interaction with Numb and SKIP suggested an important role for LNX in tumorogenesis.     

Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion

Cohesion between sister chromatids is established during S phase and maintained through G2 phase until it is resolved in anaphase (for review, see [1-3]). In Saccharomyces cerevisiae, a complex consisting of Scc1, Smc1, Smc3, and Scc3 proteins, called "cohesin," mediates the connection between sister chromatids. The evolutionary conserved yeast protein Eco1 is required for establishment of sister chromatid cohesion during S phase but not for its further maintenance during G2 or M phases or for loading the cohesin complex onto DNA. We address the molecular functions of Eco1 with sensitive sequence analytic techniques, including hidden Markov model domain fragment searches. We found a two-domain architecture with an N-terminal C2H2 Zn finger-like domain and an approximately 150 residue C-terminal domain with an apparent acetyl coenzyme A binding motif (http://mendel.imp.univie.ac.at/SEQUENCES/ECO1/). Biochemical tests confirm that Eco1 has the acetyltransferase activity in vitro. In vitro Eco1 acetylates itself and components of the cohesin complex but not histones. Thus, the establishment of cohesion between sister chromatids appears to be regulated, directly or indirectly, by a specific acetyltransferase.     

ZBP-89 enhances Bak expression and causes apoptosis in hepatocellular carcinoma cells

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.